Proteinase K Pretreatment for the Quantitative Recovery and Sensitive Detection of the Tuberculosis Biomarker Mannose-Capped Lipoarabinomannan Spiked into Human Serum

This paper reports on an investigation of an enzymaticpretreatmentprotocol using proteinase K (ProK) for the analysis of human serumsamples spiked with mannose-capped lipoarabinomannan (ManLAM). ManLAMis an antigenic biomarker found in the serum, urine, and other bodyfluids of individuals infected with tuberculosis (TB). Immunometricmeasurements of ManLAM are compromised by steric effects due to itscomplexation with high-molecular-weight components in these matricesthat interfere with its capture and/or labeling. Recent work has shownthat deproteinization of these types of samples by perchloric acidacidification or ProK digestion releases ManLAM from complexation.Releasing ManLAM greatly improves its detectability and, as a result,its utility as a TB biomarker. The work detailed herein examined howdifferent ProK reaction conditions (e.g., enzyme concentration anddigestion time and temperature) affect the recovery and detectabilityof ManLAM in human serum. As measured by enzyme-linked immunosorbentassay (ELISA), we show that using the optimal set of digestion conditionsto free ManLAM, which also yield a small, quantitatively reproduciblelevel of sample concentration, it is possible to achieve a spikedManLAM recovery of 98 +/- 13% and a limit of detection of 10 pg/mL(0.6 pM). Experiments also demonstrated that the ELISA responses measuredfor a given ManLAM concentration in serum after pretreatment werestatistically indistinguishable from those directly determined forthe same amounts of ManLAM added to an innocuous buffered solution.Possible adaptations of the digestion protocol for use in point-of-careTB testing are also briefly discussed.